RESUMEN
Lung cancer is one of the most common and deadliest cancers. Preclinical models are essential to study new therapies and combinations taking tumor genetics into account. We have established cell lines expressing the luciferase gene from lines with varied genetic backgrounds, commonly encountered in patients with pulmonary adenocarcinoma. We have characterized these lines by testing their response to multiple drugs. Thus, we have developed orthotopic preclinical mouse models of NSCLC with very high engraftment efficiency. These models allow the easy monitoring of tumor growth, particularly in response to treatment, and of tumor cells dissemination in the body. We show that concomitant treatment with osimertinib (3rd generation tyrosine kinase inhibitor targeting mutated EGFR) and bevacizumab (anti-angiogenic targeting VEGF) can have a beneficial therapeutic effect on EGFR-mutated tumors. We also show that the addition of afatinib to osimertinib-treated tumors in escape leads to tumor growth inhibition. No such effect is observed with selumetinib or simvastatin. These preclinical mouse models therefore make it possible to test innovative therapeutic combinations and are also a tool of choice for studying resistance mechanisms.
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Acrilamidas , Afatinib , Compuestos de Anilina , Bevacizumab , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Animales , Compuestos de Anilina/farmacología , Compuestos de Anilina/uso terapéutico , Acrilamidas/farmacología , Afatinib/farmacología , Afatinib/uso terapéutico , Bevacizumab/farmacología , Bevacizumab/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Ratones , Humanos , Línea Celular Tumoral , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Modelos Animales de Enfermedad , Ensayos Antitumor por Modelo de Xenoinjerto , Receptores ErbB/metabolismo , Receptores ErbB/genética , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , Quinazolinas/administración & dosificación , Piperazinas/farmacología , Piperazinas/uso terapéutico , Piperazinas/administración & dosificación , Femenino , Indoles , PirimidinasRESUMEN
The catalytically inactive caspase-8-homologous protein, c-FLIP, is a potent antiapoptotic protein highly expressed in various types of cancers. c-FLIP competes with caspase-8 for binding to the adaptor protein FADD (Fas-Associated Death Domain) following death receptors' (DRs) activation via the ligands of the TNF-R family. As a consequence, the extrinsic apoptotic signaling pathway involving DRs is inhibited. The inhibition of c-FLIP activity in tumor cells might enhance DR-mediated apoptosis and overcome immune and anticancer drug resistance. Based on an in silico approach, the aim of this work was to identify new small inhibitory molecules able to bind selectively to c-FLIP and block its anti-apoptotic activity. Using a homology 3D model of c-FLIP, an in silico screening of 1880 compounds from the NCI database (National Cancer Institute) was performed. Nine molecules were selected for in vitro assays, based on their binding affinity to c-FLIP and their high selectivity compared to caspase-8. These molecules selectively bind to the Death Effector Domain 2 (DED2) of c-FLIP. We have tested in vitro the inhibitory effect of these nine molecules using the human lung cancer cell line H1703, overexpressing c-FLIP. Our results showed that six of these newly identified compounds efficiently prevent FADD/c-FLIP interactions in a molecular pull-down assay, as well as in a DISC immunoprecipitation assay. The overexpression of c-FLIP in H1703 prevents TRAIL-mediated apoptosis; however, a combination of TRAIL with these selected molecules significantly restored TRAIL-induced cell death by rescuing caspase cleavage and activation. Altogether, our findings indicate that new inhibitory chemical molecules efficiently prevent c-FLIP recruitment into the DISC complex, thus restoring the caspase-8-dependent apoptotic cascade. These results pave the way to design new c-FLIP inhibitory molecules that may serve as anticancer agents in tumors overexpressing c-FLIP.
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Drug nanocapsules coated with iron oxide nanoparticles (SPION) were elaborated by the simultaneous nanoprecipitation of the drug and the nanoparticles, through solvent shifting. We examined four drugs: sorafenib, sorafenib tosylate, α-tocopherol and paclitaxel, to cover the cases of molecular solids, ionic solids, and molecular liquids. We first investigated the formation of the drug core in the final mixture of solvents at different concentrations. A Surfactant-Free Micro-Emulsion domain (SFME, thermodynamically stable) was observed at low drug concentration and an Ouzo domain (metastable) at high drug concentration, except for the case of paclitaxel which crystallizes at high concentration without forming an Ouzo domain. When co-nanoprecipitated with the molecular drugs in the Ouzo domain (sorafenib or α-tocopherol), the SPION limited the coalescence of the drug particles to less than 100 nm, forming capsules with a drug encapsulation efficiency of ca 80 %. In contrast, larger capsules were formed from the SFME or when using the ionic form (sorafenib tosylate). Finally, the sorafenib-SPION capsules exhibit a similar chemotherapeutic effect as the free drug on the hepatocellular carcinoma in vitro.
Asunto(s)
Neoplasias Hepáticas , Nanocápsulas , Humanos , Nanocápsulas/química , Solventes , Sorafenib , alfa-Tocoferol , Estructura Molecular , Paclitaxel , Nanopartículas Magnéticas de Óxido de HierroRESUMEN
Fistulizing anoperineal lesions are severe complications of Crohn's disease (CD) that affect quality of life with a long-term risk of anal sphincter destruction, incontinence, permanent stoma, and anal cancer. Despite several surgical procedures, they relapse in about two-thirds of patients, mandating innovative treatments. Ultrasmall particles of iron oxide (USPIO) have been described to achieve in vivo rapid healing of deep wounds in the skin and liver of rats thanks to their nanobridging capability that could be adapted to fistula treatment. Our main purpose was to highlight preclinical data with USPIO for the treatment of perianal fistulizing CD. Twenty male Sprague Dawley rats with severe 2,4,6-trinitrobenzenesulfonic acid solution (TNBS)-induced proctitis were operated to generate two perianal fistulas per rat. At day 35, two inflammatory fistulas were obtained per rat and perineal magnetic resonance imaging (MRI) was performed. After a baseline MRI, a fistula tract was randomly drawn and topically treated either with saline or with USPIO for 1 min (n = 17 for each). The rats underwent a perineal MRI on postoperative days (POD) 1, 4, and 7 and were sacrificed for pathological examination. The primary outcome was the filling or closure of the fistula tract, including the external or internal openings. USPIO treatment allowed the closure and/or filling of all the treated fistulas from its application until POD 7 in comparison with the control fistulas (23%). The treatment with USPIO was safe, permanently closed the fistula along its entire length, including internal and external orifices, and paved new avenues for the treatment of perianal fistulizing Crohn's disease.
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Enfermedad de Crohn , Fístula Rectal , Animales , Masculino , Ratas , Enfermedad de Crohn/complicaciones , Enfermedad de Crohn/tratamiento farmacológico , Enfermedad de Crohn/patología , Nanopartículas Magnéticas de Óxido de Hierro , Recurrencia Local de Neoplasia , Calidad de Vida , Ratas Sprague-Dawley , Fístula Rectal/tratamiento farmacológico , Fístula Rectal/etiología , Resultado del TratamientoRESUMEN
In recent decades, 3Din vitrocultures of primary human hepatocytes (PHHs) have been increasingly developed to establish models capable of faithfully mimicking main liver functions. The use of 3D bioprinting, capable of recreating structures composed of cells embedded in matrix with controlled microarchitectures, is an emergent key feature for tissue engineering. In this work, we used an extrusion-based system to print PHH in a methacrylated gelatin (GelMa) matrix. PHH bioprinted in GelMa rapidly organized into polarized hollow spheroids and were viable for at least 28 d of culture. These PHH were highly differentiated with maintenance of liver differentiation genes over time, as demonstrated by transcriptomic analysis and functional approaches. The cells were polarized with localization of apico/canalicular regions, and displayed activities of phase I and II biotransformation enzymes that could be regulated by inducers. Furthermore, the implantation of the bioprinted structures in mice demonstrated their capability to vascularize, and their ability to maintain human hepatic specific functions for at least 28 d was illustrated by albumin secretion and debrisoquine metabolism. This model could hold great promise for human liver tissue generation and its use in future biotechnological developments.
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Bioimpresión , Animales , Bioimpresión/métodos , Gelatina/química , Hepatocitos/metabolismo , Humanos , Hidrogeles/química , Ratones , Impresión Tridimensional , Ingeniería de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
Glioblastoma remains a cancer for which the effectiveness of treatments has shown little improvement over the last decades. For this pathology, multiple therapies combining resection, chemotherapy and radiotherapy remain the norm. In this context, the use of high-Z nanoparticles such as gold or hafnium to potentiate radiotherapy is attracting more and more attention. Here, we evaluate the potentiating effect of hollow shells made of gold and iron oxide nanoparticles (hybridosomes®) on the radiotherapy of glioblastoma, using murine GL261-Luc+ brain tumor model. While iron oxide seems to have no beneficial effect for radiotherapy, we observe a real effect of gold nanoparticles-despite their low amount-with a median survival increase of almost 20% compared to radiotherapy only and even 33% compared to the control group. Cellular and in vivo studies show that a molecule of interest nano-precipitated in the core of the hybridosomes® is released and internalized by the surrounding brain cells. Finally, in vivo studies show that hybridosomes® injected intra-tumorally are still present in the vicinity of the brain tumor more than 5â¯days after injection (duration of the Stupp protocol's radiation treatment). Interestingly, one mouse treated with radiotherapy in the presence of gold-containing hybridosomes® survived 78â¯days. Monitoring of the tumoral growth of this long-term survivor using both MRI and bioluminescence revealed a decrease of the tumor size after treatment. These very encouraging results are a proof-of-concept that hybridosomes® are really effective tools for the development of combined therapies (chemo-radiotherapy).
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Neoplasias Encefálicas , Glioblastoma , Nanopartículas del Metal , Nanocápsulas , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/radioterapia , Línea Celular Tumoral , Glioblastoma/tratamiento farmacológico , Glioblastoma/radioterapia , Oro/uso terapéutico , Nanopartículas del Metal/uso terapéutico , Ratones , Nanocápsulas/uso terapéuticoRESUMEN
BACKGROUND: Osimertinib is a third generation tyrosine kinase inhibitor (TKI) that targets the epidermal growth factor receptor (EGFR) in lung cancer. However, although this molecule is not subject to some of the resistance mechanisms observed in response to first generation TKIs, ultimately, patients relapse because of unknown resistance mechanisms. New relevant non-small cell lung cancer (NSCLC) mice models are therefore required to allow the analysis of these resistance mechanisms and to evaluate the efficacy of new therapeutic strategies. METHODS: Briefly, PC-9 cells, previously modified for luciferase expression, were injected into the tail vein of mice. Tumor implantation and longitudinal growth, almost exclusively localized in the lung, were evaluated by bioluminescence. Once established, the tumor was treated with osimertinib until tumor escape and development of bone metastases. RESULTS: Micro-metastases were detected by bioluminescence and collected for further analysis. CONCLUSION: We describe an orthotopic model of NSCLC protocol that led to lung primary tumor nesting and, after osimertinib treatment, by metastases dissemination, and that allow the isolation of these small osimertinib-resistant micro-metastases. This model provides new biological tools to study tumor progression from the establishment of a lung tumor to the generation of drug-resistant micro-metastases, mimicking the natural course of the disease in human NSCLC patients.
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Acrilamidas/uso terapéutico , Compuestos de Anilina/uso terapéutico , Neoplasias Óseas/secundario , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Micrometástasis de Neoplasia , Animales , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos BALB C , Mutación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We investigate the encapsulation in hybridosomes®, a type of capsules unique regarding their structure and method of elaboration. Hybridosomes® are made of a single shell of inorganic nanoparticles (~5 nm) crosslinked with a polymer and are easily obtained via spontaneous emulsification in a ternary mixture THF/water/butylated hydroxytoluene (BHT). Our main finding is that an exceptionally high concentration of a hydrophobic model dye can be loaded in the hybridosomes®, up to 0.35 mol.L-1 or equivalently 170 g.L-1 or 450,000 molecules/capsule. The detailed investigation of the encapsulation mechanism shows that the dye concentrates in the droplets during the emulsification step simultaneously with capsule formation. Then it precipitates inside the capsules during the course of solvent evaporation. In vitro fluorescence measurements show that the nano-precipitated cargo can be transferred from the core of the hybridosomes® to the membrane of liposomes. In vivo studies suggest that the dye diffuses through the body during several days. The released dye tends to accumulate in body-fat, while the inorganic nanoparticles remain trapped into the liver and the spleen macrophages.
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Nanocápsulas , Nanopartículas , Interacciones Hidrofóbicas e Hidrofílicas , Polímeros , SolventesRESUMEN
Epithelial ovarian cancer (EOC) represents 5% of human gynecologic cancers in the world, is heterogeneous and highly invasive with a dismal prognosis (5 year-survival rate <35%). Diagnosis of EOC is frequently made at advanced stages and, despite aggressive treatments combining surgery and chemotherapy, fatal relapse rapidly occurs and is accompanied by a peritoneal carcinosis. In this context, novel therapeutical advances are urgently required. Adoptive transfer(s) of immune effector cells, including allogeneic human Vγ9Vδ2 T lymphocytes, represent attractive targets for efficiently and safely tracking tissue-invading tumor cells and controlling tumor dissemination in the organism. Our study describes the establishment of robust and physiological orthotopic model of human EOC in mouse, that includes surgical resection (ovariectomy) and chemotherapy, which are ineluctably accompanied by a fatal peritoneal carcinosis recurrence. Through a complementary set of in vitro and in vivo experiments, we provide here a preclinical proof of interest of the antitumor efficiency of adoptive transfers of allogeneic human Vγ9Vδ2 T lymphocytes against EOC, in association with surgical debulking and standard chemotherapies (i.e., taxanes and platinum salts). Moreover, our results indicate that chemo- and immunotherapies can be combined to improve the antitumor efficiency of immunotherapeutic lines. Altogether, these results further pave the way for next-generation antitumor immunotherapies, based on local administrations of human allogeneic human Vγ9Vδ2 T lymphocytes, in association with standard treatments.
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PURPOSE: Cellular immunotherapies are currently being explored to eliminate highly invasive and chemoradioresistant glioblastoma (GBM) cells involved in rapid relapse. We recently showed that concomitant stereotactic injections of nonalloreactive allogeneic Vγ9Vδ2 T lymphocytes eradicate zoledronate-primed human GBM cells. In the present study, we investigated the spontaneous reactivity of allogeneic human Vγ9Vδ2 T lymphocytes toward primary human GBM cells, in vitro and in vivo, in the absence of any prior sensitization. EXPERIMENTAL DESIGN: Through functional and transcriptomic analyses, we extensively characterized the immunoreactivity of human Vγ9Vδ2 T lymphocytes against various primary GBM cultures directly derived from patient tumors. RESULTS: We evidenced that GBM cells displaying a mesenchymal signature are spontaneously eliminated by allogeneic human Vγ9Vδ2 T lymphocytes, a reactivity process being mediated by γδ T-cell receptor (TCR) and tightly regulated by cellular stress-associated NKG2D pathway. This led to the identification of highly reactive Vγ9Vδ2 T lymphocyte populations, independently of a specific TCR repertoire signature. Moreover, we finally provide evidence of immunotherapeutic efficacy in vivo, in the absence of any prior tumor cell sensitization. CONCLUSIONS: By identifying pathways implicated in the selective natural recognition of mesenchymal GBM cell subtypes, accounting for 30% of primary diagnosed and 60% of recurrent GBM, our results pave the way for novel targeted cellular immunotherapies.
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Glioblastoma/inmunología , Glioblastoma/patología , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/patología , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Apoptosis , Proliferación Celular , Glioblastoma/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos NOD , Ratones SCID , Pronóstico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human VÏ9Vδ2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.
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Neoplasias Encefálicas/inmunología , Glioblastoma/inmunología , Traslado Adoptivo , Adulto , Animales , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioblastoma/patología , Xenoinjertos , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Glioblastoma multiforme (GBM) remains the most frequent and deadliest primary brain tumor in adults despite aggressive treatments, because of the persistence of infiltrative and resistant tumor cells. Nonalloreactive human Vγ9Vδ2 T lymphocytes, the major peripheral γδ T-cell subset in adults, represent attractive effectors for designing immunotherapeutic strategies to track and eliminate brain tumor cells, with limited side effects. We analyzed the effects of ex vivo sensitizations of Vγ9Vδ2 T cells by IL-21, a modulating cytokine, on their cytolytic reactivity. We first showed that primary human GBM-1 cells were naturally eliminated by allogeneic Vγ9Vδ2 T lymphocytes, through a perforin/granzyme-mediated cytotoxicity. IL-21 increased both intracellular granzyme B levels and cytotoxicity of allogeneic human Vγ9Vδ2 T lymphocytes in vitro. Importantly, IL-21-enhanced cytotoxicity was rapid, which supports the development of sensitization(s) of γδ T lymphocytes before adoptive transfer, a process that avoids any deleterious effect associated with direct administrations of IL-21. Finally, we showed, for the first time, that IL-21-sensitized allogeneic Vγ9Vδ2 T cells significantly eliminated GBM tumor cells that developed in the brain after orthotopic administrations in vivo. Altogether our observations pave the way for novel efficient stereotaxic immunotherapies in GBM patients by using IL-21-sensitized allogeneic human Vγ9Vδ2 T cells.
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Neoplasias Encefálicas/inmunología , Vacunas contra el Cáncer/inmunología , Glioblastoma/inmunología , Inmunoterapia Adoptiva/métodos , Interleucinas/metabolismo , Linfocitos T/inmunología , Adulto , Animales , Neoplasias Encefálicas/terapia , Línea Celular Tumoral , Citotoxicidad Inmunológica , Glioblastoma/terapia , Humanos , Isoantígenos/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos NOD , Cultivo Primario de Células , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismoRESUMEN
BACKGROUND AIMS: To produce an anti-leukemic effect after hematopoietic stem cell transplantation we have long considered the theoretical possibility of using banks of HLA-DP specific T-cell clones transduced with a suicide gene. For that application as for any others, a clonal strategy is constrained by the population doubling (PD) potential of T cells, which has been rarely explored or exploited. METHODS: We used clinical-grade conditions and two donors who were homozygous and identical for all HLA-alleles except HLA-DP. After mixed lymphocyte culture and transduction, we obtained 14 HLA-DP-specific T-cell clones transduced with the HSV-TK suicide gene. Clones were then selected on the basis of their specificity and functional characteristics and evaluated for their doubling potential. RESULTS: After these steps of selection the clone NAT-DP4(TK), specific for HLA-DPB1*04:01/04:02, which produced high levels of interferon-γ (IFNγ), tumor necrosis factor (TNF), interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), was fully sequenced. It has two copies of the HSV-TK suicide transgene whose localizations were determined. Four billion NAT-DP4(TK) cells were frozen after 50 PDs. Thawed NAT-DP4(TK) cells retain the potential to undergo 50 additional PDs, a potential very far beyond that required to produce a biological effect. This PD potential was confirmed on 6/16 additional different T-cell clones. This type of well-defined clone can also support a second genetic modification with CAR constructs. CONCLUSION: The possibility of choosing rare donors and exploiting the natural proliferative potential of T lymphocytes may dramatically reduce the clinical and immunologic complexity of adoptive transfer protocols that rely on the use of third-party T-cell populations.
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Células Clonales/citología , Técnicas Citológicas/métodos , Cadenas beta de HLA-DP , Linfocitos T/citología , Animales , Donantes de Sangre , Genes Transgénicos Suicidas , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Cadenas beta de HLA-DP/genética , Cadenas beta de HLA-DP/inmunología , Humanos , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Prueba de Cultivo Mixto de Linfocitos , Ratones , Linfocitos T/inmunología , Timidina Quinasa/genética , Transducción Genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Purpose: Glioblastoma (GBM) is the most common and malignant form of primary human brain tumor in adults, with an average survival at diagnosis of 18 months. Metabolism is a new attractive therapeutic target in cancer; however, little is known about metabolic heterogeneity and plasticity within GBM tumors. We therefore aimed to investigate metabolic phenotyping of primary cultures in the context of molecular tumor heterogeneity to provide a proof of concept for personalized metabolic targeting of GBM.Experimental Design: We have analyzed extensively several primary GBM cultures using transcriptomics, metabolic phenotyping assays, and mitochondrial respirometry.Results: We found that metabolic phenotyping clearly identifies 2 clusters, GLNHigh and GLNLow, mainly based on metabolic plasticity and glutamine (GLN) utilization. Inhibition of glutamine metabolism slows the in vitro and in vivo growth of GLNHigh GBM cultures despite metabolic adaptation to nutrient availability, in particular by increasing pyruvate shuttling into mitochondria. Furthermore, phenotypic and molecular analyses show that highly proliferative GLNHigh cultures are CD133neg and display a mesenchymal signature in contrast to CD133pos GLNLow GBM cells.Conclusions: Our results show that metabolic phenotyping identified an essential metabolic pathway in a GBM cell subtype, and provide a proof of concept for theranostic metabolic targeting. Clin Cancer Res; 23(20); 6292-304. ©2017 AACR.
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Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Glutamina/metabolismo , Mitocondrias/metabolismo , Animales , Biomarcadores , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Análisis por Conglomerados , Biología Computacional/métodos , Modelos Animales de Enfermedad , Metabolismo Energético , Perfilación de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Glucosa/metabolismo , Xenoinjertos , Humanos , Metabolómica/métodos , Ratones , Modelos Biológicos , FenotipoRESUMEN
The inherent resistance of cancer stem cells (CSCs) to existing therapies has largely hampered the development of effective treatments for advanced malignancy. To help develop novel immunotherapy approaches that efficiently target CSCs, an experimental model allowing reliable distinction of CSCs and non-CSCs was set up to study their interaction with non-MHC-restricted γδ T cells and antigen-specific CD8+ T cells. Stable lines with characteristics of breast CSC-like cells were generated from ras-transformed human mammary epithelial (HMLER) cells as confirmed by their CD44hi CD24lo GD2+ phenotype, their mesenchymal morphology in culture and their capacity to form mammospheres under non-adherent conditions, as well as their potent tumorigenicity, self-renewal and differentiation in xenografted mice. The resistance of CSC-like cells to γδ T cells could be overcome by inhibition of farnesyl pyrophosphate synthase (FPPS) through pretreatment with zoledronate or with FPPS-targeting short hairpin RNA. γδ T cells induced upregulation of MHC class I and CD54/ICAM-1 on CSC-like cells and thereby increased the susceptibility to antigen-specific killing by CD8+ T cells. Alternatively, γδ T-cell responses could be specifically directed against CSC-like cells using the humanised anti-GD2 monoclonal antibody hu14.18K322A. Our findings identify a powerful synergism between MHC-restricted and non-MHC-restricted T cells in the eradication of cancer cells including breast CSCs. Our research suggests that novel immunotherapies may benefit from a two-pronged approach combining γδ T-cell and CD8+ T-cell targeting strategies that triggers effective innate-like and tumour-specific adaptive responses.
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Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Linfocitos T CD8-positivos/inmunología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Animales , Anticuerpos/farmacología , Mama/patología , Citotoxicidad Inmunológica , Difosfonatos/farmacología , Células Epiteliales/metabolismo , Epítopos/inmunología , Femenino , Humanos , Imidazoles/farmacología , Inmunidad Innata , Interferón gamma/metabolismo , Complejo Mayor de Histocompatibilidad , Ratones , Fenotipo , Ácido Zoledrónico , Proteínas ras/metabolismoRESUMEN
Glioblastoma multiforme (GBM) represents the most frequent and deadliest primary brain tumor. Aggressive treatment still fails to eliminate deep brain infiltrative and highly resistant tumor cells. Human Vγ9Vδ2 T cells, the major peripheral blood γδ T cell subset, react against a wide array of tumor cells and represent attractive immune effector T cells for the design of antitumor therapies. This study aims at providing a preclinical rationale for immunotherapies in GBM based on stereotaxic administration of allogeneic human Vγ9Vδ2 T cells. The feasibility and the antitumor efficacy of stereotaxic Vγ9Vδ2 T cell injections have been investigated in orthotopic GBM mice model using selected heterogeneous and invasive primary human GBM cells. Allogeneic human Vγ9Vδ2 T cells survive and patrol for several days within the brain parenchyma following adoptive transfer and can successfully eliminate infiltrative GBM primary cells. These striking observations pave the way for optimized stereotaxic antitumor immunotherapies targeting human allogeneic Vγ9Vδ2 T cells in GBM patients.
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Buruli ulcer, a debilitating disease, is caused by Mycobacterium ulcerans. The incidence of this neglected tropical disease is steadily increasing. As a rule, without treatment, skin ulcers occur and a lengthy healing process may be observed associated with severe functional disabilities. Mouse models are already available to study establishment of lesions or evaluation of therapy but a lack of a suitable animal model, mimicking all clinical stages, in particular the healing process, remains an obstacle to understand the pathophysiology of M. ulcerans infection. M. ulcerans was s.c. inoculated in three consanguine mouse strains, that is, BALB/c and C57BL/6, classically used to study mycobacterial infection, and FVB/N. Strikingly, FVB/N mice, although as sensitive as all other mouse strains with respect to M. ulcerans infection, presented a spontaneous healing after the ulcerative phase despite stable bacterial load, and mycolactone toxin was not detected in the healed tissues. The spontaneous healing process was accompanied by an activation of the innate immune system. The adaptive response initiated by FVB/N mice was not involved in the healing process and did not confer protection against M. ulcerans. Our work highlights the importance of innate immune responses to control M. ulcerans infection. This in vivo model of M. ulcerans infection now paves the way for new avenues of research toward the elucidation of critical stages of this disease, such as the characterization of the regulation of mycolactone production, a better understanding of the pathophysiology of M. ulcerans infection, and the development of new therapeutic strategies.
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Úlcera de Buruli/fisiopatología , Macrólidos/metabolismo , Mycobacterium ulcerans/inmunología , Animales , Úlcera de Buruli/microbiología , Modelos Animales de Enfermedad , Regulación Bacteriana de la Expresión Génica/inmunología , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos , Remisión Espontánea , Especificidad de la EspecieRESUMEN
Using brain lymphoma model, we demonstrate that immunotherapy combining Treg depletion (using anti-CD25 mAb PC61) followed by intracranial CpG-ODN administration induced tumor rejection in all treated mice and led to the establishment of a memory antitumor immune response in 60% of them. This protective effect was associated with a recruitment of NK cells and, to a lesser extent, of dendritic cells, B cells and T lymphocytes. NK cell depletion abolished the protective effect of the treatment, confirming a major role of NK cells in brain tumor elimination. Each treatment used alone failed to protect brain tumor bearing mice, revealing the therapeutic benefit of combining Treg depletion and local CpG-ODN injection.
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Adyuvantes Inmunológicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Linfoma/tratamiento farmacológico , Oligodesoxirribonucleótidos/uso terapéutico , Linfocitos T Reguladores/fisiología , Animales , Anticuerpos/toxicidad , Antígenos Ly/inmunología , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Neoplasias Encefálicas/complicaciones , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Citocinas/metabolismo , Modelos Animales de Enfermedad , Etopósido/farmacología , Etopósido/uso terapéutico , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Subunidad alfa del Receptor de Interleucina-2/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/fisiología , Linfoma/complicaciones , Linfoma/metabolismo , Linfoma/patología , Trastornos de la Memoria/etiología , Trastornos de la Memoria/prevención & control , Ratones , Ratones Endogámicos C57BL , Subfamilia B de Receptores Similares a Lectina de Células NK/inmunología , Trasplante de Neoplasias , Linfocitos T Reguladores/efectos de los fármacos , Factores de Tiempo , Receptor Toll-Like 9/metabolismoRESUMEN
Microglia are the major myeloid-immune cells of the brain parenchyma. In a steady state, microglia monitor their environment for pathogens or damaged cells. In response to neural injury or inflammation, microglia become competent APCs able to prime CD4(+) and CD8(+) T lymphocytes. We previously demonstrated that neonatal and adult microglia cross-present exogenous soluble Ags in vitro. However, whether microglia are able to cross-present Ag to naive CD8(+) T cells in vivo, within the brain microenvironment, remains undetermined. Here, we have designed an original protocol in order to exclude the involvement in cross-presentation activity of peripheral migrating APCs and of CNS-associated APCs. In C57Bl/6 mice, in which the body but not the head has been properly irradiated, we analyzed the ability of resident microglia to stimulate intracerebrally injected CD8(+) T cells in vivo. This study demonstrates for the first time that adult microglia cross-present Ag to naive CD8(+) T cells in vivo and that full microglia activation is required to overcome the inhibitory constrains of the brain and to render microglia able to cross-prime naive CD8(+) T cells injected in the brain. These observations offer new insights in brain-tumor immunotherapy based on the induction of cytotoxic antitumoral T cells.